Method for the production of dextran of relatively low molecular weight



Patented Nov. 24, 1953 "METHGD' FOR"THE PRODUCTIONOF DEX- TRAN OF'RELATIVELY LOW MOLECULAR WEIGHT Haroldll. Eoepsell, Henry M. Tsuchiya, and Nison NJlHllmanQPeoria,111., assignors to the United "States of America as represented. by the Secrotary of'Agricu'lture N Drawing. iApplicationMarch.11,1952, :Serial No. 276,033

(Granted-under-Title 35,'U.. S. Code (1952),

.sec. 266) 7 Claims.

A non-exclusive, irrevocable, royalty free rl-icense in the invention herein .described,.afor"all governmental purposes,throughout the world, with the power. to; grant sublicenses for suchpurposes, is hereby; granted to the Governmentof the United States of America.

This invention relatestothe, preparationof the polysaccharide, dextran. it relates;.in.particular, to the polymerization reaction whereby sucrose is utilized :as -a raw .material. to' produce a polymer of glucose in which the recurring an hydroglucose uni-ts are linked,predominately at the 1-6 positions. More particularly, it: relates to novel polymerizationmethods whereby the polymerization is carried out with the assistance of"dextrazvsyrithesizing"enz ines under conditions which inherently resultin a dextran'pro'duct oflow' molecularweight. *Stillmore particu-- larly, itrelatesfinbne'of' itsmajor phases, to carrying out the dextran synthesis in the absence of appreciable -quantities of microorganisms.

In this-specification andclaims, the generic term -dextran-refers to the polysaccharide commonly known in the art" as native "dextran, whichmay be characterizedas a'polymer of. glucose in which the recurring anhydroglucose .units are linked predominately through the 1-6 positions. "The term, as used hereimin'cludes such polymerswhich possess a molecular weight range =of from 1;000 upward to'many'millions. .Thisinventi'on relates'also in several ofl'its phases- 130 polymers -o.f molecular'weight of less -than L000, which compounds may be termed oligosac- Charities.

' Dex-tren hesgenerallyfound 'valueas av carbohydrate-gum having a'wide' variety of industrial uses. Many of its uses,.particular1y. those relatin to'blood volume expanders, require a molecular weight'range'which is lower than that found in the native 'dextran as commonly .produoed bymi'crobiological synthesis. -This high molecular Weight is characteristic, whether the nativedeatranis produced by prior whole culture synthesis wherein the dextranap-roducing organismsremain inthe' syntheshiliquors during-the formation of the 'deXtran, or. by the re cently developed enzym'ic synthesis wherein the dextran-producing enzyme (dextransucrase) l is produced in a separata'initial.step, separated from the-microorganisms, and thenemployed .for the dextran synthesis as a subsequent step. This latter method of synthesis is disclosed and claimed incopending applications Ser. No. 215,623, filed March 15, 1951,iby"Koepsell,..Kazenko, Jeanes, Sharpe and William, and .Ser. No. 256,586, filed November 15, 1951, by .-Tsucl1iya-and=Koepsell. Because-of the =very zninor proportion ofclow molecular weight dcxtran'compared with thezhigh .molecular weight dextran, .prior workers :have

.novel; control of therenvironment'of{the dextran synthesis. -We have discovered that the relative proportion of this lowwmolecular weightsdextran increases :as the initial sucrose concentration in the polymerization:mediumis increased. .'I?he:low

molecular weight producedzaccordingwto :our: in-

vention-may be recovered by simple fractionation methods -.as a, separata'eccnomically' significant product having: avariety of .uses' not possessed by the dextran of high molecular weight.

.Our low =-molecular weight-'2 dextran, whichzis novel atv least insofar as being an economically significant product obtained by direct synthesis, is characterized bya molecular weightwhichlmay vary from as low as-6,000:to as high as 400,000, the particular rangesawithin theser-limits. depending upon certain: variables :as Will be explained in detail below.

The low molecular'wveight.dextran produced by our invention is useful'as a thickeningagent, as an adhesive. as a 2protective-.=colloid, andas an intermediate in plasticsand. pharmaceuticals. It appears to possess .Lutilityi similar tothat of the various dextrins and tube-useful for many of the purposes in which :the. so called native dextran of highsmolecularlweight is now used. In addition, by virtue of its molecular 'structure, it possesses iusefulsproperties, particularly in-the lower molecular :weight ranges; not shared by either the r-dextrins; orv the native dextrans.

We efiect control overthe proportion' of the two- .dextrans,::produced by the enzyme irom-a given microbiological. source, i. e.,- the-dextran possessing alzmolecularaweight of one million or higher .and the dextran :possessing a molecular weightof 1,000 to140l),000, by controlling the concentration biz-sucrose :in' the. synthesis" medium. We have discovered that thea'propor-tion oflow molecular'weighttdextran'increases as the initial concentration. of: sucrose in the synthesis medium is increased.

.Characteristically, -the proportion of the low molecular weightuproduct isproduced ineconomically. significant amounts as the sucrose concentration-is increased to above that'norm'ally used in thepreparation of'native-dextrans. "At concentrations of 25. :to grams sucrose per mlof solutionpthe' quantity bf recoverable low molecular weight dextran becomes considerable and increases as the concentration of sucrose is increased. At concentrations of -40 grams per 100 ml., the proportion of low molecular weight dextran frequently becomes the predominating product of the synthesis, and at 60-80 grams per 100 ml. of solution the low molecular weight dextran is usually the sole product of the synthesis.

As previously mentioned, dextran may be produced enzymatically, in the absence of microorganisms. This method of dextran synthesis is important in carrying out our present invention, for it affords the production of dextran at high sucrose concentrations. Although our invention is not so limited, we prefer to carry out our novel process by the enzymic method, since much higher sucrose concentrations are operable than are tolerated by the microorganisms themselves.

The molecular weight and structure of the dextran produced according to our invention, i. e., the low molecular weight product, is afiected by the particular microorganism involved. Where enzymic synthesis methods are used, the particular molecular weight is affected by the microbiological source of the dextran-producing enzyme. The dextran-synthesizing organism used in this invention may be any organism of the genus Leuconostoc which produces dextran as a major metabolic product under the dextran synthesizing conditions familiar to the art. Within these classes of organisms, some produce, in our inventive method, a product of the low molecular weight type having a molecular weight as low as 1,000 or less. Other microorganisms result in a low molecular weight dextran having a molecular weight of 10,000 to 20,000, while still others produce a low molecular weight dextran in the higher limits as heretofore disclosed. Still another variable which is dependent upon the organism source of the enzyme is the particular structure and mode of linkage between glucopyranosyl groups in the molecule. The ratio of 1-6 to non-1-6 linkages appears to be affected by the particular organism source.

Utilizing our discoveries, we conduct dextran synthesis, preferably by the enzymic method in sucrose solutions which may vary from 25 grams per 100 ml. up to saturation with sucrose. The dextransucrase enzyme may be prepared in accordance with the disclosure of the copending applications previously acknowledged. We may use either the crude culture filtrate, or the isolated enzyme preparation as obtained in accordance with those applications. We may, in general, employ culture liquors rich in dextransucrase, as for example, those from previous dextran syntheses which actually contain the unseparated cells, or the culture of the previously mentioned copending applications before cell removal. We may also employ whole culture methods, but these methods are usually limited to sucrose solutions of about grams per 100 ml. concentration or less, depending upon the specific sucrose tolerance of the microorganism employed.

The concentration of dextransucrase employed in our invention, or the relative proportion of the enzyme with respect to the sucrose in the synthesis medium, may vary over a wide range. Increasing the amount of enzyme units usually has the effect of accelerating the polymerization.

Our invention thus affords means for producing significant quantities of low molecular weight dextran in the form of easily separable mixtures with high molecular weight dextran, both products being highly and uniquely useful. It affords, moreover, means for controlling the relative proportion of the two dextrans produced as well as means for predetermination of the character of the low molecular weight material. The former is accomplished by varying the concentration of sucrose in the polymerization medium, whereas the latter is accomplished by selection of the particular dextransucrase composition giving the required product, especially as pertains to the particular organism source of the enzyme. As previously stated, the enzyme may be prepared by the method of the noted copending application. This involves cultivation of a dextran producing organism on a lean sucrose medium until all the sucrose is used up and then recovering the enzyme by removing the cells of the organism. The culture liquor may be used directly in the polymerization process, in which case the sucrose may merely be added thereto. Alternatively, the enzyme may be recovered from the culture liquor by evaporation or precipitation and used when and as desired. The improved method of enzyme production disclosed and claimed by Tsuchiya and Koepsell in application Ser. No. 256,586 may be employed. This improvement involves conducting the enzyme synthesis within a pH range of 6.0 to 7.0 and thereafter substantially immediately altering the pH to 4.8-5.5 to avoid enzyme breakdown.

The following specific examples illustrate our invention.

EXAMPLE 1 Leuconostoc mesenieroides NRRL B-5l2 was grown in a medium containing 2 percent sucrose, 2 percent corn steep liquor solids, 0.1 percent potassium monobasic phosphate, and small amounts of nutrient salts. The pH of the medium was maintained at 6.5 to 6.8 by the addition of caustic solution as needed, and the temperature was maintained at 25 C. The medium was stirred and aerated during the fermentation. Sucrose utilization was complete within 10 hrs., and the culture was immediately adjusted to pH 5.0 and cooled overnight to minimize enzyme destruction. The next morning the culture was adjusted to pH 7.0 by the addition of alkali. This resulted in a precipitation of insoluble matter, which was removed along with the cells by filtration. The clear, amber solution was immediately adjusted to pH 5.0 for stability maintenance. Upon assay, it was found to contain 42 units of dextr-ansucrase per ml., where one unit is defined as the amount of enzyme which will convert 1 mg. of sucrose to dextran in 1 hr. under optimal conversion conditions.

A series of reaction mixtures was made up containing this culture filtrate, and varying amounts of sucrose in the range of initial concentrations of 10 to 70 grams per 100 ml. of solution, adjusted to pH 5.0, and incubated at 30 C. until conversion of sucrose to dextran, as measured by the production of fructose, had ceased. The amounts of fructose produced coincided closely to the theoretical amounts corresponding with the weight of sucrose taken. In the series of flasks, a marked difference in opalescence and especially viscosity was noted, the flasks containing the higher proportion of sucrose exhibiting the lesser degrees of the phenomena.

The crude dextran product was obtained from the reaction mixtures by adding alcohol to give an percent solution. The precipitate was of a gummy character, and tended to produce insoluble, aggregated masses. Separation of the 5.. lextra was: a complished. by c ntric-li ation. The crude extran was; then suspend d n water with autoclav ne. to duce. an: essentiallyolorless: s lution. The ov rall. yield. or lextran: was d termined by pt cal rotat on. on; the. basis that purifieddextran possesse a. specific; rotation of, 5- This solution. was. diluted to a. 2.-pe.1:- t solution. d. 51x11. aliquotsweretreated with absolute, e hanol. to. ive the.- results described. in Table I b low. This. cumulative. alcohol free:- tionation, as shown in the table, gives a, distriution of; th extrans pr duced arrangedrin c r anc with heir. espective.- molecular weights; the higher molecular weight product bfi nggpliey. cipitated by the lower concentration; of ethanol. Exam natienoi th abledisclose th t the dextrans. pr duced. an d ff rentiated. into. two maior-groupson the basis or molecularsize. The first to:Precipitat, lcohol concentrations. up. to 42, per n is. hew-calle na iv hi h m lecularweight dextran. The second, that which precipitates t l ohol concentration reater. than 42 percent. is the lower molecular weight small dextran. It is also seen that the size of, the small dextran component differed in the, reaction mixtures, and that small dextran pro duced at 70 grams of. sucrose per 100 ml. of solution, which failed to precipitate at less than 60 percent. alcohol, was appreciably smaller than the small dextran produced at grams of sucrose per 100 m1... of solution which precipitated at alcohol concentrations in the range of 50 to 80, percent alcohol.

Additional portions of the individual reaction mixtures were precipitated with alcohol at. '75 percent to obtain the dextran product as described above. High molecular weight dextran was removed by precipitation at 42 percent alcohol, and the small dextran was obtained by precipitation from the supernatant liquid at 75 percent. alcohol. The molecular weight of the small dextran was determined by light scattering methods. The analysis indicated that the average molecular weight of, the small dextran pro duced at 30 percent sucrose was about, 24,000, while that produced at 50 percent sucrose was about 10,000. The latter small dextran product was divided into two portions on the basis of solubility or insolubility at 55 percent alcohol.

By viscosity measurements the soluble portion had" a. molecular weight below 10,000, while the insoluble portion had a molecular weight of about 13,000.

The small dextran, precipitating above 42 percent alcohol, was identified as being a dextran by the characteristic infra-red absorption spectrum of dextran. The small size of this dextran was also manifested by the low viscosity and lack of opalescence of its solutions.

Mg. dextran precipitated 1 Sucrose, g./l00 ml.

1 100 mg. dextran were taken.

The dextran-synthesizingreaction mixture was prepared at grams of sucrose per 100ml. of solution as described in Example 1. Such reaction mixtures have a pronounced tendency to produce insoluble aggregated masses of dextran upon prolonged standing. A reaction mixture in which this process had proceeded to such an extent that the solution contained a large volume of curdlike aggregate was used as a source of the small dextran. The solution (750 ml.) was,

thinned to a workable consistency with 40 percent alcohol and theaggregated masses of dex- Table II.-CumuZative alcohol precipitation of 3-512 demtran from aggregated synthesis mixture Percent I Mg. dcxtrau alcohol I precipitated 100 mg. dextran were taken.

EXAMPLE 3 Lcuconostoc mesenteroz'des NRRL B4072 was grown in a medium containing 2 percent sucrose, 2 percent corn steep liquor solids, 2 percent monobasic potassium phosphate, and small,

amounts of nutrient salts. During production of the enzyme, the pH of the culture remained in. the region from 6.0 to 7.0 by virtue of the buffering capacity of the phosphate. Upon utilization of all of the sucrose, the culture was centrifuged to remove the bacterial cells, and the culture, filtrate was adjusted to pH 5.2 for stability maintenance.

A series of. dextran-producing reaction mixtures, varying from 30 to 7.0 grams sucrose. per

100ml. of solution, was set. up as described in.

Example 1; The. dextransprecipitation procedure, leading; to the obtaining of cumulative alcohol precipitation curves, was performed asv described in Table I. The data obtained are shown in Table III.

Examination of this table verifies the production of both small and large dextran components, as seen in Table I, and it is again evident that the average molecular size of the small component increased as the initial sucrose concentration of the reaction mixture decreased.

7 Table III.Cumulative alcohol precipitation of 3-1072 dextran obtained at various sucrose concentrations 2 k dcxtran precipitated l 100 mg. (lest-run were when.

EXAMPLE 4 Leuconostoc mesenteroides NRRL B-523, which usually produces a water-insoluble dextran, was grown in the medium described. in Example 3.

The culture filtrate obtained after removal of cells had a potency of 30 dextransucrase units per ml.

A series of reaction mixtures containing sucross in the range of to 70 grams per 100 ml. was set up as in Example 1. During the course of the reaction it was noted that none of the flocs of the insoluble-type of dextran ordinarily produced by this strain of microorganism was produced in the flasks containing 60 to '70 grams sucrose per 100 ml. The flocs appeared after the second day in only small quantity in reaction mixtures containing 40 to 50 grams sucrose per 100 ml., and were noted soon after the beginning of the reaction and in considerably heavier amounts at lower sucrose levels, time necessary for completion of the conversion, as evidenced by fructose measurements, varied from 22 hrs. at 10 percent sucrose to 234 hours at 70 grams sucrose per 100 m1, and the amounts of reducing sugars, as determined by the method of Somogyi, Jour. Biol. Chem. 160, 61 (1945), were about to percent greater than those calculated from the amounts of sucrose involved.

The dextran was precipitated from the reaction mixtures at 90 percent alcohol. The total yield of dextran varied from 12 to 23 percent of the theoretical calculated from the sucrose used. Cumulative alcohol fractionations of the watersoluble portion of the dextran showed that relatively small molecular weight polymer predominated. Most of this portion precipitated in each case at alcohol concentrations of percent and greater, and the dextrans produced at sucrose levels of to grams per 100 mi. required alcohol concentrations of above percent to effect precipitation. The dextran product, when reprecipitated, had an infra-red absorption spectrum typical of dextrans.

We claim:

1. The method comprising subjecting an aqueous solution of sucrose having a concentration within the range of 25 grams to 75 grams per m1. of solution to the action of a dextransucrase enzyme, thereby to cause synthesis of deXtran of a relatively low molecular weight and recovering said low molecular weight dextran from the reaction medium.

The length of .4

2. The method comprising subjecting sucrose in aqueous solution to the action of dextransucrase, the concentration of sucrose in said medium being within the range of 25 grams to 75 grams per 100 ml. of solution, thereby to cause synthesis of dextran of a relatively low molecular weight and recovering said low molecular weight dextran from the reaction medium.

3. Method of claim 2 in which the dextran of low molecular weight is recovered by the addition of ethanol to the reaction medium.

4. The method of claim 2 in which the dextran produced possesses a molecular weight within the range of 1,000 to 400,000.

5. The method comprising adding sucrose to an aqueous reaction medium containing dextransucrase to produce a solution containing 25-75 grams sucrose per 100 ml. solution thereby to cause synthesis of dextran of a relatively low molecular weight, separating high molecular weight native dextran from said solution when present by the addition of a low molecular weight alkanol, removing the precipitated native dextran and recovering said low molecular weight dextran from the reaction medium by the addition of more low molecular weight all-:anol.

6. The method of claim 4 wherein the concentration of sucrose is about 70 grams per 100 ml. and the reaction medium permitted to stand until the dextran forms a curd-like aggregate, and separating the aggregate by thinning with alcohol and centrifuging.

l. The method comprising adding sucrose to an aqueous reaction medium containing dextransucrase to produce a solution containing in-- itially 25-75 grams of sucrose per 100 ml. solution, thereby to cause synthesis of dextran of a relatively low molecular weight, separating high molecular weight native dextran from said solution by the addition of a low molecular weight alkanol added to bring the alkanol concentration to approximately 50 percent, removing the precipitated native dextran and recovering said low melecular weight dextran from the reaction medium by the addition of more low molecular weight alkanol.

HAROLD J. KOEPSELL. HENRY M. TSUC'l-IIYA. NISON N. HELLMAN.

References Cited in the file of this patent UNITED STATES PATENTS Name Date Owen Jan. 1, 1946 OTHER REFERENCES Number 

1. THE METHOD COMPRISING SUBJECTING AN AQUEOUS SOLUTION OF SUCROSE HAVING A CONCENTRATION WITHIN THE RANGE OF 25 GRAMS TO 75 GRAMS PER 100 ML, OF SOLUTION TO THE ACTION OF A DEXTRANSUCRASE ENZYME, THEREBY TO CAUSE SYNTHESIS OF DEXTRAN OF A RELATIVELY LOW MOLECULAR WEIGHT AND RECOVERING SAID LOW MOLECULAR WEIGHT DEXTRAN FROM THE REACTION MEDIUM. 